The labeling of proteins with fluorescent substituents, most commonly done using green fluorescent protein (GFP) and its derivatives is an invaluable tool for tagging and visualizing proteins in vivo. The ability to genetically encode protein labels as with GFP combined with the superior characteristics of small molecule fluorophores will significantly improve the in vivo tagging and visualization of proteins. This proposal aims to exploit the high-affinity interaction between Mtx and DHFR to label proteins in vivo by fusing the protein of interest to DHFR and then labeling the protein with small-molecule Mtx conjugates. In Preliminary Results, we present work recently submitted for publication demonstrating the feasibility of this approach, labeling both a plasma membrane and a nuclear protein fused to DHFR with Mtx-Texas Red in Chinese Hamster Ovary (CHO) cells. We then outline our plans to develop orthogonal Mtx-DHFR variants via protein engineering in Aim 1. In Aim 2, the modular Mtx-DHFR labeling approach is applied to CALl to distinguish between the role of myosin-llb localized to the cell posterior to that localized to the lamella in cell motility, an experiment difficult to achieve with current labeling methodologies. Finally, in Aim 3 we demonstrate the use of Mtx-DHFR labels for multicolor tagging and FRET applications and characterize the efficacy of these labels in comparison to GFP. [unreadable] [unreadable] [unreadable]